The present invention relates to the cloning of the gene encoding a 28.5 kDa Toxoplasma excretion-secretion antigen, and to the production of peptide fragments representing epitopes thereof, as well as to preparations of said antigen and of its fragments, and to their uses.
Toxoplasmosis is one of the most widespread protozoal infections, both in man and in animals. It is responsible for about 25% of deaths in AIDS patients. The congenital infection, the cause of abortions or severe neonatal malformations in man and domestic animals, could be prevented. Indeed, it is known that the primary infection induces a long-lasting immunity.
In the search for protective antigens permitting the development of a vaccine against toxoplasmosis, various antigens have been studied. The inventors"" team was in particular interested in the excretion-secretion antigens (ESA) of the tachyzoites. It has indeed been established, during experiments both in man and in animals, that the ESA antigens were immunogenic. It was also shown that some ESAs possess epitopes in common with antigens of bradyzoites. However, bradyzoites are the resistant form of the parasite.
Various approaches, comprising in particular the production of monoclonal antibodies, colloidal gold labeling, and molecular biology, led the inventors"" team to the characterization of four common antigens [CHARIF et al. Exp. Parasitol., 71:117 (1990)], [CESBRON-DELAUW et al., Proc. Natl. Acad. Sci. USA, 86:7537 (1989)], [DARCY et al., Parasitol. Res. 76:478, (1990)]. The main one, called Gra2 or GP28.5, is a glycoprotein of 28.5 kDa, and it has been shown that it is a constituent of the matrix of the dense granules of tachyzoites, and that it is associated with the microvilli network of the parasitophorus vacuole of the parasite, after invasion of the host.
A 28 kDa antigen (P28), considered as being similar to the GP28.5 antigen, has been described by SIBLEY and SHARMA [SIBLEY et al. Infect. Immun. 55:2137 (1987)], and a DNA sequence encoding this antigen has been published by PRINCE et al. [Molec. Biochem. Parasitol. 34:3 (1989)].
The present invention set itself the aim of producing purified preparations of the GP28.5 antigen, as well as of producing this antigen in recombinant form, and its immunological characterization. In particular, the aim of the present invention is the localization and the characterization of specific epitopes of the GP28.5 antigen.
The inventors succeeded in obtaining a purified preparation of the GP28.5 antigen, and demonstrated the protective effect of an immunization by this preparation against Toxoplasma gondii infection in mice.
The inventors also cloned the entire gene encoding the GP28.5 antigen, and located the introns and exons, as well as the 5xe2x80x2 and 3xe2x80x2 noncoding regions.
The subject of the present invention is a nucleic acid fragment, which comprises a sequence encoding the Toxoplasma GP28.5 antigen. A nucleic acid fragment conforming to the invention is represented in the list of sequences in the annex under the number SEQ.ID.NO:1. xe2x80x9cSequence encoding the Toxoplasma GP28.5 antigenxe2x80x9d is understood to mean not only the coding sequence identified in the sequence SEQ.ID.NO:1, but also any sequence which, taking into account the degeneracy of the genetic code, encodes the polypeptide represented in the list of sequences in the annex under the number SEQ.ID.NO:2.
The inventors also showed that the GP28.5 antigen contains several major epitopes specific for the B cells; one of them, which is recognized by a mouse monoclonal antibody called TG17-179 [CHARIF et al., Exp. Parasitol., 71:117 (1990)] is located at the C-terminal end of the molecule.
The inventors characterized this epitope and showed that it contained at least the 5 C-terminal amino acids of GP28.5. In addition, they showed that this epitope is also a major epitope recognized by human polyclonal antibodies directed against T. gondii. 
The invention also encompasses nucleic acid fragments which encode polypeptides representing epitopes of the GP28.5 antigen.
According to a preferred embodiment of the present invention, said nucleic acid fragment encodes a polypeptide comprising at least the 5 C-terminal amino acids of the sequence SEQ.ID.NO:2.
According to another preferred embodiment of the invention, said nucleic acid fragment encodes a polypeptide comprising fragment 24-129 of the sequence SEQ.ID.NO:2.
According to yet another preferred embodiment of the invention, said nucleic acid fragment encodes a polypeptide comprising fragment 127-176 of the sequence SEQ.ID.NO:2.
The subject of the invention is also recombinant vectors (plasmids, viruses and the like), which comprise at least one nucleic acid fragment as defined above, encoding the Toxoplasma GP28.5 antigen, or a peptide fragment representing an epitope thereof.
Said vectors are in particular expression vectors, comprising sequences of the promoter type, terminator type and the like.
The subject of the present invention is also transformed eukaryotic or prokaryotic cells (and in particular microorganisms), which contain at least one recombinant vector in accordance with the invention.
The subject of the invention is also a polypeptide of 185 amino acids whose sequence which is represented in the list of sequences in the annex under the number SEQ.ID.NO:2, is that of the Toxoplasma GP28.5 antigen.
The invention also encompasses the polypeptides whose sequence differs from the abovementioned sequence only by a few amino acids, in particular polypeptides representing allelic variants or isoforms of the GP28.5 antigen.
The invention also comprises recombinant proteins comprising all or part of the sequence of the SEQ.ID.NO:2 sequence, optionally fused with another polypeptide sequence; within this framework, the subject of the invention is in particular:
a recombinant protein of 212 amino acids, comprising the entire sequence of the GP28.5 antigen;
a recombinant protein comprising amino acids 127-176 of the sequence SEQ.ID.NO:2;
a recombinant protein comprising amino acids 1-129 of the sequence SEQ.ID.NO:2;
a recombinant protein comprising amino acids 24-129 of the sequence SEQ.ID.NO:2.
The invention also relates to a process for producing a recombinant protein as defined above, which process comprises a step during which the transformed cells as defined above, comprising at least one DNA fragment in accordance with the invention, are cultured.
The recombinant polypeptides in accordance with the invention, when expressed in E. coli, conserve major epitopes involved in the polyclonal response to the GP28.5 antigen, and do so even though the expression in E. coli does not maintain the structural integrity of the GP28.5 protein which is a glycosylated protein.
If it is desired, however, to obtain other epitopes such as epitopes of carbohydrate nature, or epitopes corresponding to tertiary and quaternary structures, it will be chosen to produce the recombinant GP28.5 in eukaryotic systems such as for example yeasts or baculoviruses.
The inventors, in addition, showed that the C-terminal sequence of fifteen amino acids of the GP28.5 antigen represents an epitope recognized by a number of sera from patients suffering from acute or chronic infections, and also located other major B epitopes in the fragment corresponding to amino acids 127-176 of the sequence SEQ.ID.NO:2. In addition, the inventors located other regions between amino acids 24 and 129, and in particular the regions corresponding to amino acids 55-70 and to amino acids 140-160 of the sequence SEQ.ID.NO:2, which comprise epitopes of the GP28.5 antigen which are recognized by the human sera.
The subject of the invention is also peptides comprising at least one epitope of the GP28.5 antigen.
According to a preferred embodiment of the invention, the said peptide comprises the 5 C-terminal amino acids of the sequence SEQ.ID.NO:2.
According to a preferred arrangement of this embodiment, the said peptide comprises between 5 and 15 C-terminal amino acids of the sequence SEQ.ID.NO:2.
According to a preferred feature of this arrangement, said peptide comprises the 8 C-terminal amino acids of the sequence SEQ.ID.NO:2.
According to another preferred embodiment of the invention, said peptide comprises amino acids 24-129 of the sequence SEQ.ID.NO:2.
According to another preferred embodiment of the invention, said peptide comprises amino acids 55 to 70 of the sequence SEQ.ID.NO:2.
According to yet another preferred embodiment of the invention, said peptide comprises amino acids 140 to 160 of the sequence SEQ.ID.NO:2.
The sequence of the 5 C-terminal amino acids of the GP28.5 antigen is recognized in immunotransfer by the mouse monoclonal antibody TG 17-179. A competitive ELISA with longer peptides has shown that the immunoreactivity was conserved for peptides of 8 residues or more, and lost when the peptide was reduced to the last 6 C-terminal residues or less. Experiments with the octapeptide lacking the C-terminal glutamine residue showed that it was then 20 times less active. On the other hand, neither the addition of residues to the C-terminal end, nor the substitution of the terminal COOH functional group change the immunoreactivity of the epitope. In addition, competition experiments between the monoclonal antibody TG 17-179 and sera from infected patients showed that the epitope defined by this monoclonal antibody is also a major epitope for the human polyclonal antibodies.
The subject of the invention is also antigenic compositions which comprise at least one antigen chosen from the group consisting of:
a purified preparation of GP28.5 antigen;
a polypeptide of sequence SEQ.ID.NO:2;
a fragment of said polypeptide representing an epitope of the GP28.5 antigen;
a recombinant protein comprising all or part of the sequence SEQ.ID.NO:2.
The invention also encompasses a process for preparing polyclonal or monoclonal anti-GP28.5 antibodies which comprises a step during which an animal is immunized with an antigenic composition in accordance with the invention.
The antigenic compositions in accordance with the invention also permit the preparation of diagnostic reagents or anti-Toxoplasma vaccines. The invention also encompasses these reagents and these vaccines.
The present invention will be understood more clearly with the aid of the additional description below, which refers to examples relating to the cloning of the gene encoding the GP28.5 antigen of Toxoplasma gondii, and the identification of the specific epitopes of this antigen.